Citations

Background Geenius HIV 1/2 Supplemental Assay (Geenius; Bio-Rad Laboratories) is the only Food and Drug Administration–approved HIV-1/HIV-2 antibody differentiation test for the second step in the HIV laboratory testing algorithm. We characterized the occurrence of true HIV-1 and HIV-2 infections as well as false results in 6 US clinical laboratories using Geenius. Methods We examined routine HIV testing outcome data from the time the laboratories began using the algorithm with Geenius until September 30, 2017. We calculated the positive predictive value for Geenius HIV-1 and HIV-2 reactivity separately. Results Of 5,046,684 specimens tested, 41,791 had reactive antigen/antibody test results. Most specimens with reactive antigen/antibody results were HIV-1 antibody–positive established infections (n = 32,421), 1,865 of which also had indeterminate HIV-2 bands present. Ninety-three specimens were HIV-2 antibody positive or untypable for HIV-1/HIV-2 antibody. Acute HIV-1 infections were found in 528 specimens; 881 specimens lacked the nucleic acid test to determine the possibility of acute HIV-1 infection. False-positive antigen/antibody test results were present in 7505 specimens. Few specimens (n = 363) had false-positive antigen/antibody results with indeterminate Geenius and negative HIV-1 nucleic acid test results. The positive predictive values of Geenius reactivity were 99.4% for HIV-1 and 4.3% for HIV-2. Conclusions Routine testing using the laboratory testing algorithm with Geenius resulted in most specimens resolving as HIV negative or HIV-1 positive. The occurrence of indeterminate HIV-2 bands with a Geenius final assay interpretation of HIV-1 positive was more common than true HIV-2 infections. Reporting indeterminate HIV-2 results in this situation may cause confusion with interpreting HIV infection status.

Abstract Background Quantitative nucleic acid amplification (QNAT) tests are cornerstone for the management of CMV disease after organ transplantation. We assessed the potential impact of viral load results obtained by two commercial WHO international standard-calibrated NAT in solid-organ (SOT) and hematopoietic stem cell transplant (HSCT) recipients. Methods Sixty-four patients (36 SOT and 28 HSCT) had plasma CMV viral load assessed using the COBAS AmpliPrep/COBAS TaqMan CMV Test (CAP/CTM; lower limit of quantification [LLoQ] at 137 IU/mL) and cobas 6800 System (cobas CMV; LLoQ at 35 IU/mL). Viral load values were correlated with clinical course and outcomes. Results Forty-three of 64 patients (67.2%) had CMV infection or disease (asymptomatic, 67.4%; gastrointestinal disease, 16.3%; pneumonitis 4.7%) at median of 4.4 months (IQR 1.4 to 7.7) from transplantation. At CMV infection diagnosis, viral load results (mean ± SD) were almost two-fold higher when measured by cobas CMV (19,456 ± 51,618 IU/mL) compared with CAP/CTM (10,504 ± 27,744 IU/mL; P = 0.04). Time to onset of CMV viremia was significantly shorter (11.5 days; P < 0.001) while viral clearance was significantly longer (12.75 days; P < 0.001) by cobas CMV when compared with CAP/CTM. Persistent viremia was observed with cobas CMV in 44% of patients at the time of first negative results by CAP/CTM. Patients with negative results by cobas CMV at the end of antiviral treatment had a significantly lower need for re-treatment (OR 0.26, 95% CI 0.04 to 0.99, P = 0.05). Conclusion Our study highlights significant differences between CMV QNAT assays despite calibration to the WHO-international standard. The significant differences in the degree (almost two-fold), time to onset (12 days difference) and clearance (13 days difference) of CMV viremia between two automated commercial QNAT assays have direct implications in the care of transplant recipients. Persistence of low-level viremia was observed in samples that reached negative threshold by CAP/CTM, when tested using the more sensitive cobas CMV. Clearance of CMV viremia, when assessed by the more sensitive cobas CMV, was significantly associated with a lower need for re-treatment. Disclosures All authors: No reported disclosures.

AbstractCytomegalovirus (CMV) viral loads overall were 0.29 log IU/mL higher with cobas CMV for use on the cobas 6800/8800 System (cobas CMV) compared with Cobas AmpliPrep/Cobas TaqMan CMV Test (CAP/CTM CMV). Cytomegalovirus DNAemia was detected 11.5 days earlier by cobas CMV, whereas clearance was delayed by 12.8 days. Cytomegalovirus remained detectable by cobas CMV in 44.2% of patients at the time of viral clearance as determined by CAP/CTM CMV. Undetectable viral load by cobas CMV at end of treatment was associated with reduced risk for retreatment (odds ratio, 0.26; 95% confidence interval, 0.04–0.99; P = .05).The use of different quantitative cytomegalovirus nucleic acid tests may affect direct patient care as a result of significant differences in reporting the degree of CMV DNAemia and the time to first detection and clearance of CMV DNAemia.

Abstract The critical nature of the microbiology laboratory in infectious disease diagnosis calls for a close, positive working relationship between the physician/advanced practice provider and the microbiologists who provide enormous value to the healthcare team. This document, developed by experts in laboratory and adult and pediatric clinical medicine, provides information on which tests are valuable and in which contexts, and on tests that add little or no value for diagnostic decisions. This document presents a system-based approach rather than specimen-based approach, and includes bloodstream and cardiovascular system infections, central nervous system infections, ocular infections, soft tissue infections of the head and neck, upper and lower respiratory infections, infections of the gastrointestinal tract, intra-abdominal infections, bone and joint infections, urinary tract infections, genital infections, and other skin and soft tissue infections; or into etiologic agent groups, including arthropod-borne infections, viral syndromes, and blood and tissue parasite infections. Each section contains introductory concepts, a summary of key points, and detailed tables that list suspected agents; the most reliable tests to order; the samples (and volumes) to collect in order of preference; specimen transport devices, procedures, times, and temperatures; and detailed notes on specific issues regarding the test methods, such as when tests are likely to require a specialized laboratory or have prolonged turnaround times. In addition, the pediatric needs of specimen management are also emphasized. There is intentional redundancy among the tables and sections, as many agents and assay choices overlap. The document is intended to serve as a guidance for physicians in choosing tests that will aid them to quickly and accurately diagnose infectious diseases in their patients.

Abstract The critical nature of the microbiology laboratory in infectious disease diagnosis calls for a close, positive working relationship between the physician/advanced practice provider and the microbiologists who provide enormous value to the healthcare team. This document, developed by experts in laboratory and adult and pediatric clinical medicine, provides information on which tests are valuable and in which contexts, and on tests that add little or no value for diagnostic decisions. This document presents a system-based approach rather than specimen-based approach, and includes bloodstream and cardiovascular system infections, central nervous system infections, ocular infections, soft tissue infections of the head and neck, upper and lower respiratory infections, infections of the gastrointestinal tract, intra-abdominal infections, bone and joint infections, urinary tract infections, genital infections, and other skin and soft tissue infections; or into etiologic agent groups, including arthropod-borne infections, viral syndromes, and blood and tissue parasite infections. Each section contains introductory concepts, a summary of key points, and detailed tables that list suspected agents; the most reliable tests to order; the samples (and volumes) to collect in order of preference; specimen transport devices, procedures, times, and temperatures; and detailed notes on specific issues regarding the test methods, such as when tests are likely to require a specialized laboratory or have prolonged turnaround times. In addition, the pediatric needs of specimen management are also emphasized. There is intentional redundancy among the tables and sections, as many agents and assay choices overlap. The document is intended to serve as a guidance for physicians in choosing tests that will aid them to quickly and accurately diagnose infectious diseases in their patients.

AbstractCytomegalovirus (CMV) pneumonia causes major morbidity and mortality. Its diagnosis requires demonstration of viral cytopathic changes in tissue, entailing risks of lung biopsy. This study aimed to determine CMV viral load (VL) thresholds in bronchoalveolar lavage fluid (BALF) for diagnosis of CMV pneumonia in immunocompromised patients. CMV VL in BALF was studied in 17 patients (83% transplant recipients) and 21 control subjects with and without CMV pneumonia, respectively, using an FDA‐approved PCR assay (Cobas® AmpliPrep/Cobas TaqMan® CMV Test, Roche Molecular Systems, Inc.) calibrated to the WHO International Standard for CMV DNA (NIBSC: 09/162). Receiver operating characteristic curve analysis produced a BALF CMV VL threshold of 34 800, IU/mL with 91.7% sensitivity and 100.0% specificity for diagnosis of possible, probable, and proven CMV pneumonia in transplant patients, while a threshold of 656 000 IU/mL yielded 100% sensitivity and specificity among biopsy‐proven cases. For all immunocompromised patients, a VL threshold of 274 IU/mL was selected. VL thresholds also were normalized to BALF cell count yielding a threshold of 0.32 IU/106 cells with 91.7% sensitivity and 90.5% specificity for possible, probable, and proven CMV pneumonia in transplant recipients. Monitoring CMV VL in BALF may be a less invasive method for diagnosing CMV pneumonia in immunocompromised patients.

Abstract Background Quantification of cytomegalovirus (CMV) deoxyribonucleic acid (DNA) has important diagnostic, prognostic, and therapeutic implications in the management of transplant recipients. We aimed to assess a viral load in plasma and whole blood that distinguishes CMV disease from asymptomatic infection in a cohort of solid organ and hematopoietic stem cell transplantation. Methods We prospectively measured and compared CMV viral load in paired plasma and whole blood samples collected from transplant recipients with CMV infection and disease. Cytomegalovirus viral loads were determined by a commercially available US Food and Drug Administration-approved quantitative assay (COBAS AmpliPrep/COBAS TaqMan CMV Test [CAP/CTM CMV]) calibrated to the first World Health Organization International Standard for CMV DNA quantification. Results Moderate agreement of CMV viral load was observed between plasma and whole blood, with 31% of samples having discordant findings, particularly among samples with low DNA levels. Among the subset of samples where both paired samples had quantifiable levels, we observed a systematic bias that reflected higher viral load in whole blood compared with plasma. Based on receiver operating curve analysis, an initial plasma CMV viral load threshold of 1700 IU/mL in solid organ transplant recipients (sensitivity 80%, specificity 74%) and 1350 IU/mL in allogeneic hematopoietic stem cell transplant recipients (sensitivity 87%, specificity 87%) distinguished CMV disease and asymptomatic infection. Conclusions This study identifies standardized viral load thresholds that distinguish CMV disease from asymptomatic infection using CAP/CTM CMV assay. We propose these thresholds as potential triggers to be evaluated in prospective studies of preemptive therapy. Plasma was better than whole blood for measuring viral load using the CAP/CTM CMV assay.

ABSTRACT Hepatitis E virus (HEV) has emerged as a cause of chronic hepatitis among immunocompromised patients. Molecular assays have become important tools for the diagnosis and management of these chronically infected patients. A real-time reverse transcription-quantitative PCR (RT-qPCR) assay utilizing Pleiades probe chemistry and an RNA internal control for the simultaneous detection and quantification of HEV RNA in human serum was developed based on an adaptation of a previously described and broadly reactive primer set targeting the overlapping open reading frame 2/3 (ORF2/3) nucleotide sequence of HEV. A chimeric bovine viral diarrhea virus construct containing an HEV RNA insert (SynTura HEV) was developed, value assigned with the first World Health Organization (WHO) international standard for HEV RNA (code 6329/10), and used to prepare working assay calibrators and controls, which supported an assay quantification range of 100 to 5,000,000 IU/ml. The analytical sensitivity (95% detection rate) of this assay was 25.2 IU/ml (95% confidence interval [CI], 19.2 to 44.1 IU/ml). The assay successfully amplified 16 different HEV sequences with significant nucleotide mismatching in primer/probe binding regions, while evaluation of a WHO international reference panel for HEV genotypes (code 8578/13) showed viral load results falling within the result ranges generated by WHO collaborative study participants for all panel members (genotypes 1 to 4). Broadly reactive RT-qPCR primers targeting HEV ORF2/3 were successfully adapted for use in an assay based on Pleiades probe chemistry. The availability of secondary standards calibrated to the WHO HEV international standard can improve the standardization and performance of assays for the detection and quantification of HEV RNA.